Passive Immunization of Chickens with Anti-Enterobactin Egg Yolk Powder for Campylobacter Control

Abstract

Enterobactin (Ent) is a highly conserved and important siderophore for the growth of many Gram-negative bacterial pathogens. Therefore, targeting Ent for developing innovative intervention strategies has attracted substantial research interest in recent years. Recently, we developed a novel Ent conjugate vaccine that has been demonstrated to be effective for controlling Gram-negative pathogens using both in vitro and in vivosystems. In particular, active immunization of chickens with the Ent conjugate vaccine elicited strong immune responses and significantly reduced intestinal colonization of Campylobacter jejuni, the leading foodborne bacterial pathogen. Given that hyperimmune egg yolk immunoglobulin Y (IgY) has been increasingly recognized as a promising and practical non-antibiotic approach for passive immune protection against pathogens in livestock, in this study, we assessed the efficacy of oral administration of broiler chickens with the anti-Ent hyperimmune egg yolk powder to control C. jejuni colonization in the intestine. However, supplementation of feed with 2% (w/w) of anti-Ent egg yolk powder failed to reduce C. jejuni colonization when compared to the control group. Consistent with this finding, the ELISA titers of the specific IgY in cecum, ileum, duodenum, gizzard, and serum contents were similar between the two groups throughout the trial. Chicken intestinal microbiota also did not change in response to the egg yolk powder treatment. Subsequently, to examine ex vivo stability of the egg yolk IgY, the chicken gizzard and duodenum contents from two independent sources were spiked with the egg yolk antibodies, incubated at 42 °C for different lengths of time, and subjected to ELISA analysis. The specific IgY titers were dramatically decreased in gizzard contents (up to 2048-fold) but were not changed in duodenum contents. Collectively, oral administration of broiler chickens with the anti-Ent egg yolk powder failed to confer protection against intestinal colonization of C. jejuni, which was due to instability of the IgY in gizzard contents as demonstrated by both in vivo and ex vivo evidence.

Ex Vivo Evaluation of Egg Yolk IgY Degradation in Chicken Gastrointestinal Tract

“…Consistent with this finding, immunoblotting using specific anti-IgY antibodies clearly indicated that the IgY was undetected after 30 or 60 min of treatment in gizzard content, while its integrity was still maintained well in the matrix of control saline or HCl upon 60 min of treatment ( Figure 2C ). These data are also consistent with previous ELISA analysis of IgY titer upon acid treatment ( 7 ) and provide further direct evidence demonstrating that acidic condition alone (e.g., pH 3.0) did not alter the IgY integrity.…”

“…The above dynamic assessment of IgY stability in gizzard content confirmed our previous in vivo and ex vivo findings ( 7 ) and suggested enzymatic degradation of IgY in gizzard. To obtain direct evidence of IgY degradation, we subsequently conducted SDS-PAGE and immunoblotting assays on the egg yolk IgY upon different treatments.…”

“…In our recent study (7), the pooled small intestine content did not affect the titer of specific IgY with up to 2.5 h of treatment, suggesting weak or lack of IgY-degrading capability of small intestine content. In this study, using SDS-PAGE and immunoblotting analyses, we further evaluated the IgY-degrading capacity of each small intestine content from five 6-week-old chickens.…”

“…In a recent chicken trial ( 7 ), we have observed that orally administered hyperimmune egg yolk IgY was not stable in chicken GI tract, likely due to significant enzymatic degradation in chicken gizzard, which was supported by enzyme-linked immunosorbent assay (ELISA) analysis of specific IgY in both in vivo and ex vivo samples. In this study, to better understand the magnitude and dynamics of instability of egg yolk IgY in the GI tract and obtain direct evidence of IgY degradation, fresh GI contents were collected from five individual chickens at each sampling age (2, 4, or 6 weeks), slightly processed with saline, and subsequently subjected to comprehensive ex vivo analyses using multiple approaches [i.e., ELISA, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting].…”

“…The ex vivo IgY stability assays were performed as described previously ( 5 , 7 ) with modifications. Because supplementation of lyophilized egg yolk powder in feed is the most practical and widely adopted approach for oral administration of egg yolk IgY to control microbial infections in food animals ( 1 ), the lyophilized hyperimmune egg yolk powder produced in our recent study ( 7 ) was utilized for ex vivo assays to assess IgY stability in GI contents. The egg yolk powder was reconstituted in saline at the ratio of 1:3 (w/w), followed by centrifugation at 13,000 g for 2 min.…”